Project Summary Advancing age is associated with numerous changes in gastrointestinal physiology and function which can influence the amount and type of nutrients delivered to the small intestine and colon. These changes, along with altered immune function, can affect the composition and function of the gut microbiota. However, the role of these changes on the impaired glucose homeostasis observed with aging is unknown. Interestingly, gut microbiota-generated trimethylamine N-oxide (TMAO), a metabolite of bacterial choline metabolism, increases with advancing age in both mice and humans and the latter is believed to be a consequence of age-related gut dysbiosis. Indeed, there is an increase in the abundance of trimethylamine-producing bacteria with aging. The overall objective of this R21 proposal is to establish proof-of-concept for a greater impairment in glucose homeostasis following increases in gut microbiota-generated TMAO in older compared with young adults in order to conduct a larger, more comprehensive and mechanistic trial in the future. To this end, following a two-week lead-in controlled diet, 20 healthy young adults (18-34 yrs) and 20 healthy older (65- 79 yrs) adults will complete a 4-week randomized crossover trial of both choline bitartrate (1000 mg/d) or placebo. A 2- week washout period with continued controlled feeding will separate the treatments. All subjects will be fed an isocaloric diet (50% carbohydrate, 35% fat,15% protein) controlled for fiber and micronutrient content for the duration of the study to avoid the potential confound of individual differences in diet on gut microbiota. Measurements of insulin sensitivity and beta cell function via IVGTT, glucose tolerance via mixed meal tolerance test, and 24-hr glycemic control using continuous glucose monitoring, TMAO concentration by UPLC-MS/MS, inflammatory cytokines via ELISA, and gut microbiota composition/function using 16S rRNA pyrosequencing and targeted qt-PCR, respectively will be made before and following each 4-week treatment period. The findings from this integrative physiological study have significant translational potential as they may advance basic science findings in rodents to humans and provide novel mechanistic insight into observational studies in humans by establishing a specific role of the gut microbiome in modulating insulin sensitivity and 24-hr glycemic control with aging. In turn, the gut microbiota may be a key target for therapies that contribute to the maintenance of normal glucose homeostasis with aging. Importantly, our study may provide rationale for individualizing nutritional or pharmaceutical interventions that target the gut microbiota as an interface between the food we eat and host physiology.